Regulating Divergent Transcriptomes through mRNA Splicing

International Journal of

Molecular Sciences

Review

Regulating Divergent Transcriptomes through mRNASplicing and Its Modulation Using VariousSmall Compounds

Ken-ichi Fujita, Takaki Ishizuka, Mizuki Mitsukawa, Masashi Kurata and Seiji Masuda *

Division of Integrated Life Sciences, Graduate School of Biostudies, Kyoto University, Kyoto 606-8502, Japan;[email protected] (K.-i.F.); [email protected] (T.I.);[email protected] (M.M.); [email protected] (M.K.)* Correspondence: [email protected]; Tel.: +81-75-753-9430

Received: 14 February 2020; Accepted: 12 March 2020; Published: 16 March 2020��

Abstract: Human transcriptomes are more divergent than genes and contribute to the sophisticationof life. This divergence is derived from various isoforms arising from alternative splicing. Inaddition, alternative splicing regulated by spliceosomal factors and RNA structures, such as the RNAG-quadruplex, is important not only for isoform diversity but also for regulating gene expression.Therefore, abnormal splicing leads to serious diseases such as cancer and neurodegenerative disorders.In the first part of this review, we describe the regulation of divergent transcriptomes using alternativemRNA splicing. In the second part, we present the relationship between the disruption of splicingand diseases. Recently, various compounds with splicing inhibitor activity were established. Thesesplicing inhibitors are recognized as a biological tool to investigate the molecular mechanism ofsplicing and as a potential therapeutic agent for cancer treatment. Food-derived compounds withsimilar functions were found and are expected to exhibit anticancer effects. In the final part, we describethe compounds that modulate the messenger RNA (mRNA) splicing process and their availability forbasic research and future clinical potential.

Keywords: alternative mRNA splicing; cancer; neurodegenerative disorder; splicing inhibitor;food-derived compound

1. Introduction

In eukaryotic cells, protein-coding genes are transcribed as pre-messenger RNA (mRNA) inthe nucleus and pre-mRNA undergoes several RNA processing steps, such as 5′-capping, splicing,and 3′-end processing. These gene expression processes are tightly coordinated with each other toachieve efficient and accurate gene expression [1]. After the mRNA processing steps, the maturemRNA is exported from the nucleus to the cytoplasm for translation. Human transcriptomes aremore divergent than genes. This divergence is derived from various isoforms arising from alternativesplicing, which is an essential biological process for considerable proteomic diversity and complexitydespite the relatively limited number of human genes [2]. Furthermore, alternative splicing hasimportant roles not only for expressing proteins through transcript diversity but also for regulatinggene expression. Transcripts from most human protein-coding genes undergo one or more formsof alternative splicing. Alternatively spliced isoforms vary greatly from tissue to tissue [3]. Recentcomprehensive analysis suggested that more than 40% of genes express multiple isoforms in a singletissue [4]. In particular, many isoforms containing alternative exons are expressed in neurons; thus, thebrain displays the most complex pattern of alternative splicing [5]. Alternative splicing contributes tocell differentiation and lineage determination, tissue identity acquisition and maintenance, and organ

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development [6–8]. Thus, alternative splicing is considered to be a key mechanism for regulating geneexpression networks, as well as for human diversity or sophistication.

The spliceosome, a multimegadalton ribonucleoprotein (RNP) complex comprising five snRNPsand numerous proteins, carries out splicing of pre-mRNA molecules to remove introns, then conjoinsexons in different arrangements that potentially encode alternative protein isoforms [9]. Spliceosomecomplexes are assembled at the splice sites in a pre-mRNA transcript, involving a stepwise assemblypathway of the U1, U2, and U4/5/6 snRNP spliceosome subunits. The U1 snRNP binds the 5′ splice site(5′ ss), and splicing factor 1 (SF1) and U2 snRNP auxiliary factor (U2AF) 1/2 bind the branch point (BP),polypyrimidine tract, and the 3′ ss, respectively (Figure 1). After binding, the U2 snRNP containingthe splicing factor 3A (SF3A) and splicing factor 3B (SF3B) subcomplex stably associates with BP,following the engagement of U4/U6 and U5 snRNPs in the form of a tri-snRNP particle. This leads tothe destabilization of the U1 and U4 snRNPs. These conformational and compositional rearrangementsof spliceosomal components result in the activated spliceosome; this emergence triggers two sequentialtransesterification reactions to produce the spliced-mRNA. Additional interactions that contribute tothe recognition of intron–exon boundaries and/or the spliceosome assembly are mediated by elementsof the cis-acting exonic-splicing enhancer (ESE) and intronic-splicing enhancer, and exonic-splicingsilencer (ESS) and intronic-splicing silencer, which are recognized by auxiliary splicing factors,including the Ser/Arg-rich (SR) proteins (hereafter described as SRSFs) and heterogeneous nuclearribonucleoproteins (hnRNPs) [10]. Strict recognition of the splicing site by these factors enablesindividual alternative splicing.

Splicing contributes to precise gene regulation in connection with other forms of processing in thenucleus. For example, splicing of first introns feeds back to transcription elongation and the efficiency oflast intron removal affects cleavage and polyadenylation of mRNAs [11–13]. Coupling mRNA splicingto mRNA export ensures efficient nuclear export of mature mRNPs for translation in the cytoplasmmediated by the evolutionarily conserved transcription and export (TREX) complex [1,14]. The TREXcomplex is recruited to mRNA in a splicing-dependent manner via splicing factor and UAP56, and ittriggers the association of nuclear RNA export factor 1 (NXF1), which is a final mRNA export factor,onto the export competent mRNA. Recently, a molecular mechanism that suppresses the recruitment ofNXF1 to incompletely spliced mRNAs was partly demonstrated [15–17]. In addition, gene regulationthrough nonsense-mediated mRNA decay (NMD) was shown [18–24].The aberrant transcript with thepre-mature termination codon, derived from abnormal splicing, is removed by NMD.

Mis-regulation of complicated alternative splicing is associated with cancers, and abnormalexpression or mutations in splicing factors contribute to tumorigenesis and neurodegenerativedisorders [25]. Recently, various compounds with splicing inhibitor activity were established. Thesechemical compounds are expected to act not only as a biological tool to investigate complicated splicingprocesses but also for anti-cancer drugs targeted to the splicing machinery. Food-derived compoundshaving similar functions were also identified and are receiving attention. In this review, we summarizethese compounds and discuss their potential validity in physiological function.

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undergo phase separation and assemble into membraneless organelles and fibrillar-like structures [29,34,35]. Alternative splicing events within corresponding mRNA that encode these IDR regions are significantly enriched in members of the hnRNP A and D families, which have diverse roles in splicing and other RNA biological processes. These alternative splicing events arose in mammals through evolution, and they are expected to play an important role in controlling splice site recognition by the hnRNP A and D families [36].

Figure 1. Regulation of gene expression through alternative splicing. (A) Five types of alternative splicing events. "Cassette exons" means inclusion or skipping of an exon. (B) Outline of the alternative splicing control by various factors. The U1 ribonucleoprotein (snRNP) function to recognize the 5′ splice site (ss) and the cryptic polyadenylation signals (PASs) with cleavage and polyadenylation factors (CPAFs). Splicing factor 1 (SF1), U2 snRNP auxiliary factor 1 (U2AF1), and U2AF2 bind the branch point (BP; shown as a blue “A”), polypyrimidine tract (shown as “Py-tract”), and 3′ ss (shown as a blue “AG”), respectively. U2 snRNP containing splicing factor 3B1 (SF3B1), SURP and G-patch domain containing 1 (SUGP1) displaces SF1 and binds to the BP. SR protein families and hnRNPs recognize the exonic-splicing enhancer (ESE) and exonic-splicing silencer(ESS) elements, respectively, and contribute to the recognition of the splice site and the spliceosome assembly by splicing factors. The cryptic 3′ ss (shown as a red “AG”) recognition is repressed by hnRNP. RNA G-quadruplex (rG4) structures affect splicing by acting as RNA-binding protein motifs. (C) Association of alternative splicing and gene regulation. Without U1 snRNP, the CPAF recognizes intronic PASs and generates short transcripts due to premature cleavage and polyadenylation. Some intron-containing transcripts are associated with U1 snRNP, U2AF2, and SR proteins, and they are tethered in the nucleus. Intron-containing transcripts, which are exported to the cytoplasm, often contain a premature termination codon (PTC) and are eliminated by nonsense-

Figure 1. Regulation of gene expression through alternative splicing. (A) Five types of alternativesplicing events. "Cassette exons" means inclusion or skipping of an exon. (B) Outline of the alternativesplicing control by various factors. The U1 ribonucleoprotein (snRNP) function to recognize the 5′

splice site (ss) and the cryptic polyadenylation signals (PASs) with cleavage and polyadenylation factors(CPAFs). Splicing factor 1 (SF1), U2 snRNP auxiliary factor 1 (U2AF1), and U2AF2 bind the branchpoint (BP; shown as a blue “A”), polypyrimidine tract (shown as “Py-tract”), and 3′ ss (shown as ablue “AG”), respectively. U2 snRNP containing splicing factor 3B1 (SF3B1), SURP and G-patch domaincontaining 1 (SUGP1) displaces SF1 and binds to the BP. SR protein families and hnRNPs recognize theexonic-splicing enhancer (ESE) and exonic-splicing silencer(ESS) elements, respectively, and contributeto the recognition of the splice site and the spliceosome assembly by splicing factors. The cryptic 3′ ss(shown as a red “AG”) recognition is repressed by hnRNP. RNA G-quadruplex (rG4) structures affectsplicing by acting as RNA-binding protein motifs. (C) Association of alternative splicing and generegulation. Without U1 snRNP, the CPAF recognizes intronic PASs and generates short transcripts dueto premature cleavage and polyadenylation. Some intron-containing transcripts are associated with U1snRNP, U2AF2, and SR proteins, and they are tethered in the nucleus. Intron-containing transcripts,which are exported to the cytoplasm, often contain a premature termination codon (PTC) and areeliminated by nonsense-mediated messenger RNA (mRNA) decay (NMD). Similarly, other alternativeand cryptic-spliced transcripts with PTC are also degraded by NMD.

2. Understanding the Diversity of Transcriptomes by Controlling mRNA Splicing

Alternative splicing is typically classified into five types (Figure 1A): (1) inclusion or skipping ofindividual “cassette” exons, (2) switching between alternative the 5′ and 3′ ss, (3) differential retentionof introns (RI), (4) mutually exclusive splicing of adjacent exons, and (5) alternative splicing coupledwith alternative first or last exons [3,26].

Alteration of alternatively spliced isoforms often results in changes in translational efficiency andprotein isoforms that exhibit a different enzymatic activity, location, and protein–protein interaction.

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Thus, alternative splicing contributes to the diversity of protein function [27,28]. In particular,the intrinsically disordered region (IDR), also referred to as the low-complexity region, producedby alterative splicing, attracted attention in recent studies [29,30]. The IDR is characteristic forprotein–protein interaction and regulated by post-translational modifications. The role of alternativesplicing in diversifying protein interaction capabilities was reported because region encoding of theIDR is often controlled by alternative splicing [31–33]. The IDR, which contains the abundant GY motif,was implicated in the formation of higher-order protein complexes that can undergo phase separationand assemble into membraneless organelles and fibrillar-like structures [29,34,35]. Alternative splicingevents within corresponding mRNA that encode these IDR regions are significantly enriched inmembers of the hnRNP A and D families, which have diverse roles in splicing and other RNA biologicalprocesses. These alternative splicing events arose in mammals through evolution, and they are expectedto play an important role in controlling splice site recognition by the hnRNP A and D families [36].

2.1. Relationship between Alternative SPLICING and NMD and mRNA Localization

Alternatively spliced isoforms enable distinct regulatory properties in the cell, such as individualcell mRNA stability and localization (Figure 1B,C) [37]. Regarding cell mRNA stability, the mechanismfor eliminating mRNA, which alters the reading frame, is NMD. NMD is an evolutionarily conservedcellular quality control mechanism that inspects a premature termination codon (PTC) introduced bythe change in the reading frames on the mRNA. After recognizing the PTCs, mRNA containing PTC iscleaved and eliminated from the transcriptome. PTCs are particularly problematic because they oftenresult in the production of nonfunctional and/or dominant-negative proteins [18,19].

Living organisms also regulate gene expression by efficiently using the NMD mechanism.For example, SRSF3 is a member of the SR protein family that strictly regulates its own expressionby controlling the inclusion of PTC-containing cassette exons in its own transcript using alternativesplicing. This is referred to as a “poison cassette exon” because it leads to transcript degradation byNMD [20]. Not only SRSF3 but all species in the SR protein families control the regulation of theirown expression through ultraconserved poison cassette exons [21]. These features are also observed insome RNA-binding proteins (RBPs), including the hnRNP family [22–24].

The RI attracted attention in recent years because it was demonstrated that introns contribute tothe regulation of gene expression, nuclear mRNA export, and the production of new isoforms [38].RI products can be roughly classified into two types. One class is exported to the cytoplasm withoutretention in the nucleus. This class often contains PTCs, which are frequently observed in genes encodingsplicing factors and RBPs and generally serve to downregulate protein expression by irreversiblyeliminating the PTC-containing mRNAs [39–41]. In some cases, these transcripts without PTC undergoa translation process. These were identified by ribosomal-profiling analysis and characterized by shortintrons in the 5′ UTR region, as well as enrichment of genes involved in the cell cycle [42]. The otherclass is characterized as an intron-containing transcript stably retained in the nucleus and was distinctlydefined as the detained intron (DI) by Boutz and colleagues [41]. The DI products are insensitive toNMD, and they negatively affect protein expression by preventing the respective mRNAs from beingtranslated because they are retained in the nucleus. Surprisingly, various stimuli, such as DNA damageor neuronal activation, trigger the rapid post-transcriptional splicing of the DI. As a result, the splicedDI product is immediately exported and undergoes translation [41,43–46]. In neuronal cells, genesassociated with neuronal activation tend to be long, and their transcriptional regulation is insufficientfor acute phase expression because it takes time to make a full-length transcript. Thus, divergenttranscripts with the DI synthetized from corresponding genes are pooled in the nucleus beforehand.They are spliced and exported to the cytoplasm in response to a stimulus [46]. These findings suggestthat the DI has an important role in acute phase gene expression.

Incompletely spliced mRNAs are retained within nuclear speckles (NS) in mammalian cells [17].NS are membraneless nuclear domains enriched in mRNA splicing factors, 3′ processing factors, andexport factors, and they are located in the interchromatin regions of the nucleoplasm. Recent studies

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suggested that NS act as a hub to coordinate all nuclear mRNA processing steps and quality controlsteps [47]. How mRNPs gain export capacity and how they remain in the nucleus are current areasof active research. The two mechanisms for nuclear retention are expected to be (1) active anchoringwithin the nucleus and (2) prevention of export factors being recruited.

Early spliceosomal components, U1 snRNP and U2AF2 and SR proteins, are reported to beassociated with nuclear retention [17]. In one study, depletion of U1 snRNP protein component,U1-70K, and U2AF2 prevented nuclear retention of unspliced human β-globin reporter transcripts andcaused their leakage into the cytoplasm [48]. Tethering of U1-70K and U2AF2 reporter transcripts alsocaused nuclear retention via their RS domain, which is rich in arginine and serine repeats. In addition,depletion of U2 snRNA or specific subunits of the SF3B complex did not cause this prevention,indicating that binding of the U2 snRNP is not required for nuclear retention. Moreover, it was shownthat unspliced polyadenylated RNAs that accumulate within NS were still associated with stalledinactive spliceosomes [49]. SR proteins generally have an RS domain that can be phosphorylated atmultiple positions. The phosphorylation status of the SR protein is regulated by SR protein kinaseand cdc2-like kinase families, and protein phosphatase 1 contributes to nuclear retention and splicingregulation activities [15,16,50]. Phosphorylated SR proteins are associated with splicing sites tofacilitate splicing and are dephosphorylated during splicing. After completion of splicing, SR proteinsare again phosphorylated to be recycled for the next splicing. Some SR proteins, in addition to theTREX complex, support mRNA export only in their dephosphorylated state as a result of productivesplicing [15–17]. Therefore, in the case of an incomplete splicing condition, phosphorylated SR proteinsassociated with the splicing site act as retention factors that do not recruit NXF1 for export (Figure 1C).These spliceosomal components commonly contain IDR regions with the RS domain, which suggeststhat the regulation of protein–protein interaction through the IDR region plays an important role inmRNA retention in NS. Some intron-containing transcripts are efficiently exported to the cytoplasmbecause they directly recruit NXF1 and override nuclear retention [51]. Consistently, a subset ofintron-containing cellular transcripts bound by NXF1 and SR proteins are stably detectable in thecytoplasm [52]. These findings suggest that some of the intron-containing transcripts are efficientlyexported by recruiting NXF1 and SR proteins and by escaping from retention in the nucleus.

2.2. Cryptic Splicing

The eukaryotic genome has a large number of cryptic splice sites that are rarely used under thenormal conditions but can be potentially recognized. They are often flanked by a high density ofcorresponding motifs that bind to hnRNPs and some RBPs to repress their splicing recognition [30,53].For example, hnRNP C can suppress cryptic exon recognition by binding to U-tracts in Alu elementswithin the intron [54]. NOVA alternative splicing regulator (Nova), a neuron-specific splicing factor,represses splicing by binding to long clusters of YCAY motifs [55]. TAR DNA-binging protein43 (TDP-43) and polypyrimidine tract-binding protein (PTBP)1/2, which are RBPs with variousmolecular functions related to RNA metabolism, and hnRNP L act as repressors by binding to specificrepeats [56–58]. RBM17 is implicated in cancer and a neurodegenerative disease, and it can represscryptic splice site recognition [59]. The recognition sequence of RBM17 overlaps with the targetsequence of the cryptic exon suppressed by TDP-43. U2 snRNP is also believed to be involved in therepression of cryptic splice site recognition [60–64]. These potential but inactive splice sites can berecognized when an authentic strong splice site is mutated or when there are defects in the hnRNPsand some RBPs [59]. The breakdown of the retention mechanism is linked to cancer and neuronaldiseases. We discuss these linkages in the next section.

2.3. Telescripting by U1-CPAFs

To prevent abnormal mRNA synthesis by mis-splicing, “telescripting” was identified, whichis accomplished by the unique role of U1 snRNPs in the central regulation of splicing and 3′ endcleavage/polyadenylation. It was reported that inhibition of the U1 snRNP caused not only splicing

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inhibition but also premature cleavage and polyadenylation in numerous pre-mRNAs at crypticpolyadenylation signals (PASs), frequently found in introns adjacent to the transcription start site(less than 0.5 kilobases) [65,66]. This event was not observed in U2 snRNP inhibition, and it wasrevealed that the U1 snRNP combined with intronic PASs form the complex of the U1 snRNP withcleavage and polyadenylation factors (U1-CPAFs), which are distinct from U1-spliceosomal complexesbecause of the lack of essential splicing factors [67]. U1-CPAFs co-transcriptionally protect pre-mRNAsfrom premature cleavage and polyadenylation (PCPA) at cryptic PASs in introns, thus ensuringtranscriptome integrity (Figure 1C). This function is termed “telescripting” and is separate from the rolein splicing [65,66]. In addition, U1 telescripting determines mRNA length and confers transcriptionaldirectionality from bidirectional promoters [66,68–70]. The U1 snRNP is abundantly expressedcompared with other snRNPs in human. These findings reveal a critical splicing-independent functionof U1 snRNP.

2.4. Splicing Regulation by RNA G-Quadruplex

RNA secondary structures were shown to play key roles in gene expression through regulatingvarious forms of mRNA metabolism [71,72]. Regarding the regulation of mRNA metabolism, onestable nucleic acid structure is the G-quadruplex, which is formed within guanine-rich sequences. Thisunique structure can be formed within a single strand or between multiple strands of RNA or DNA,where four G-tracts of two or more guanines, separated by short stretches of other nucleotides, areassembled in layered loops bound together through Hoogsteen hydrogen bonding. G-quadruplexeswere initially thought to focus on DNA, but they were recently found on RNA molecules as well [73].Initially, several studies demonstrated that the RNA G-quadruplex (rG4) structures in 5′ UTRs act asregulatory elements for translation [74–76]. rG4 in 5′UTRs of mRNAs, such as NRAS, KRAS, TRF2,FGF2, and VEGF can impair both cap-dependent and cap-independent translation [76–80]. Recently, itwas reported that the regulation of rG4 folding by the cytoplasmic RNA helicase DHX36 was associatedwith translational efficiency [73,81].

rG4 was also reported to be significant in regulating nuclear mRNA processing, such as 3′ endprocessing [82–84], mRNA localization [85], and alternative splicing. Indeed, several studies providedexperimental evidence that rG4 structures forming sequences proximal to the splice sites in intronsaffect the splicing and expression patterns of Bcl-xL, FMR1, and TP53 in human [86–88]. In additionto known linear RNA-binding motifs, rG4 was found to serve as an RNA-binding protein motif tomediate RNA processing. To elucidate the splicing control by rG4, research on the identification offactors recognizing rG4 is also been progressing. For example, the rG4 structure sequesters in hnRNPH, resulting in the local depletion of hnRNP H and, thus, disruption of hnRNP H-dependent splicingevents occurs [89]. In addition, it was reported that alternative splicing regulation via the rG4 structuremay control cellular processes that are important for tumor progression. hnRNP F regulates the CD44isoform switch in a rG4-dependent manner, which is associated with an epithelial–mesenchymaltransition and plays integral roles in normal development and cancer metastasis [90,91]. Profiling ofrG4 revealed widespread and evolutionarily conserved rG4 structures in the human transcriptome [92].The relationship between the splicing factor recognition of rG4 and regulation of alternative splicing isrequired for detailed research to elucidate the exact role of rG4 in alternative splicing.

3. Diseases Associated with Aberrant mRNA Splicing

Aberrant mRNA processing is an important causative factor in various diseases. AberrantmRNA splicing underlies a growing number of human diseases, including inherited disorders, cancer,diabetes, and neurodegenerative diseases [10]. Aberrant RNA splicing is caused by mutation of thetrans-acting mRNA splicing factor and the cis-element, which is an essential sequence for the bindingof splicing regulatory proteins and trans-acting mRNA splicing factor. Previous studies revealedthat a relationship between genetic diseases with abnormal splicing is associated with mutations incis-element and trans-acting factors [25]. Abnormalities in core constituents of spliceosome formation

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also underlie a discrete set of diseases, including neurodegenerative disorders and cancer. In thissection, we discuss the interaction between mRNA splicing factor mutations and disease.

3.1. Mutation of Spliceosomal Components and Cancer

Cancer has several typical characteristics, such as abnormal proliferation and alterations in cellularmetabolism. The acquisition of these features is driven by changes in gene expression [93]. It wasreported that gene regulation disorder caused by mRNA splicing factor mutation is linked to theprogression of various cancers.

Frequent and recurrent mutations are found within the early components of the RNA splicingmachinery. For example, mutations in SF3B1, U2AF1, SRSF2, and zinc finger CCCH-type, RNA-bindingmotif and serine/arginine-rich 2 (ZRSR2) were found in a variety of hematological malignancies, includingmyelodysplastic syndromes (MDSs) and chronic lymphocytic leukemia (CLL), and they are mutuallyexclusive [94]. Heterozygous hotspot missense mutation was common characteristic for SF3B1, SRSF2,and U2AF1 (Figure 2A). Mutations in ZRSR2 throughout the coding sequence caused loss-of-functionmutations [95]. Similar mutations were reported with a lower frequency in solid tumors [96]. Recently,recurrent hotspot mutations at the third nucleotide of U1 snRNA were found in several cancer types,including in medulloblastoma, with high frequency [97]. Further investigation revealed that hotspotU1 mutations were present in about 50% of sonic hedgehog (SHH) medulloblastomas, which representsone group of medulloblastomas [98]. In addition, mutations were not present across other subgroups ofmedulloblastoma, indicating that U1 snRNA mutations are highly recurrent in and extremely specificto SHH medulloblastoma. It was reported that 119 splicing factor genes carry putative driver mutationsin one or more cancer types from tumor cohort studies [99]. These reports suggested that spliceosomalmutations were considered a new hallmark and driver of tumorigenesis rather than merely passengermutations [1]. Mutations of various mRNA splicing factors were globally analyzed and shown toaffect gene expression. In addition, cancer-specific splicing changes are increasingly recognized ascontributing to tumorigenesis via various mechanisms.

3.1.1. SF3B1

SF3B1 is a member of the SF3B complex within the U2 snRNP and plays a pivotal role in theearly stages of spliceosome assembly and BP recognition [100]. Hotspot mutations in SF3B1’s HEATdomains were reported in many tumor types. These mutations induced the cryptic 3′ ss usagecurrently recognized as the most frequent splicing alteration [60]. These SF3B1 mutants are calledchange-of-function mutants because SF3B1 knockdown or overexpression does not reproduce theseforms of aberrant splicing [61]. Nearly half of the aberrant mRNA transcripts are degraded by NMD,resulting in the downregulation of gene expression [60]. There are several reports on the splicing controlmechanism by SF3B1 mutants. Mutant SF3B1 preferentially recognizes alternative BPs upstream of thecanonical BP(s), which results in deregulated usage of an alternative 3′ ss being weakly dependent onU2AF1 [61]. Because SF3B1 mutation did not alter the SF3B1–U2AF complex formation and affinitywith RNA [101], U2AF1 hotspot mutations described later did not lead to the same aberrant splicingphenotype, indicating that cryptic 3′ ss usage was specifically induced by SF3B1 mutants.

Structural analysis of the SF3B1 complex revealed that SF3B1’s HEAT domain was importantfor multiple contacts with the BP-binding proteins [101]. However, it was unknown how SF3B1mutations affect the protein interactions in the spliceosome because hotspot mutations did not affectthe stability of the SF3B1–U2AF complex and the affinity with RNA. Recently, it was reported thathotspot mutations in SF3B1 specifically disrupted the interaction with the spliceosomal protein, SURPand the G-patch domain-containing 1 (SUGP1), without the interference of other SF3B1-associatedproteins [64]. SUGP1, previously known as splicing factor 4 [102], has two tandem SURP domains anda G-patch domain. SURP domains interact with SF1, and G-patch domains were shown to activate RNAhelicases for ATP hydrolysis. Both domains are required for BP recognition by the SF3B1-containingU2 snRNP [103–105]. These findings strongly suggest that SUGP1 is involved in the BP recognition

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process. In fact, knockdown of SUGP1, but not any other members of SF3B1-associated proteins,recapitulated the aberrant splicing induced by mutant SF3B1, indicating that SUGP1 acts as a splicingregulator contributing to aberrant splicing. Those studies suggested the model shown in Figure 2Bfor SUGP1 function. SUGP1 is important for accurate BP recognition by the U2 snRNP with SF3B1,SF1, and U2AF. SUGP1 associates with and activates an unknown RNA helicase required for thedisplacement of SF1 [103], allowing base pairing between the canonical BP and U2 snRNP. Furthermore,SUGP1 overexpression partially rescued the splicing abnormalities induced by mutant SF3B1. Severalmechanisms of abnormal splicing by SF3B1 were proposed [60,61,106], implying that the structuralchanges of SF3B1 induced by mutations are substantial and need to be solved. Those reports suggestedthat the understanding of how SUGP1 restores the assembly of the mutant spliceosome can be used todevelop a potential cure for mutant SF3B1-driven cancers.


[60,61,106], implying that the structural changes of SF3B1 induced by mutations are substantial and need to be solved. Those reports suggested that the understanding of how SUGP1 restores the assembly of the mutant spliceosome can be used to develop a potential cure for mutant SF3B1-driven cancers.

Figure 2. Mutations in splicing factors and their impact on splicing. (A) Alteration of splicing caused by splicing factor mutations are shown in the boxes. U1 snRNP: purple, SF3B1: green, U2AF1: orange, Ser/Arg-rich (SR) protein 2 (SRSF2): pink, zinc finger CCCH-type, RNA-binding motif and serine/arginine-rich 2 (ZRSR2): black. Mutations in U1 snRNA cause an alteration in the splicing pattern from the canonical 5′ ss to a slightly different 5′ ss. SF3B1 mutation induces cryptic 3′ ss usage (shown as a red “AG”) and enhances intron removal. U2AF1 mutations frequently alter the usage of cassette exons. SRSF2 mutations enhance the greater binding affinity to CCwG than to GGwG in ESE, which are equally recognized by wild-type SRSF2. ZRSR2 mutations induce aberrant retention of U12-type introns. (B) Recognition of the cryptic 3′ ss induced by the mutation of SF3B1. Under normal conditions, U2 snRNP containing wild-type (WT) SF3B1 associates with SUGP1, displaces SF1 by activating RNA helicases and uses a canonical BP and 3′ ss (shown as a blue “A” and “AG”) for splicing. By contrast, U2 snRNP containing SF3B1 mutants disrupt the association with SUGP1, resulting in the use of upstream BP and cryptic 3′ ss (shown as a red “A” and “AG”) for splicing.

In addition to cryptic 3′ ss usage, widespread reduction of intron-retaining isoforms was also frequently identified in mutated SF3B1 samples [63]. The mutant SF3B1-associated decrease in intron retention was not due to the activated degradation of intron-retaining transcripts by NMD but was caused by the enhanced splicing of retained introns. The effect of this abnormality on cells is not yet known. To understand the relationship between SF3B1 abnormalities and tumorigenesis, studies on the pleiotropic splicing abnormalities are necessary.

Figure 2. Mutations in splicing factors and their impact on splicing. (A) Alteration of splicing causedby splicing factor mutations are shown in the boxes. U1 snRNP: purple, SF3B1: green, U2AF1:orange, Ser/Arg-rich (SR) protein 2 (SRSF2): pink, zinc finger CCCH-type, RNA-binding motif andserine/arginine-rich 2 (ZRSR2): black. Mutations in U1 snRNA cause an alteration in the splicingpattern from the canonical 5′ ss to a slightly different 5′ ss. SF3B1 mutation induces cryptic 3′ ss usage(shown as a red “AG”) and enhances intron removal. U2AF1 mutations frequently alter the usageof cassette exons. SRSF2 mutations enhance the greater binding affinity to CCwG than to GGwG inESE, which are equally recognized by wild-type SRSF2. ZRSR2 mutations induce aberrant retentionof U12-type introns. (B) Recognition of the cryptic 3′ ss induced by the mutation of SF3B1. Undernormal conditions, U2 snRNP containing wild-type (WT) SF3B1 associates with SUGP1, displacesSF1 by activating RNA helicases and uses a canonical BP and 3′ ss (shown as a blue “A” and “AG”)for splicing. By contrast, U2 snRNP containing SF3B1 mutants disrupt the association with SUGP1,resulting in the use of upstream BP and cryptic 3′ ss (shown as a red “A” and “AG”) for splicing.

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In addition to cryptic 3′ ss usage, widespread reduction of intron-retaining isoforms was alsofrequently identified in mutated SF3B1 samples [63]. The mutant SF3B1-associated decrease in intronretention was not due to the activated degradation of intron-retaining transcripts by NMD but wascaused by the enhanced splicing of retained introns. The effect of this abnormality on cells is not yetknown. To understand the relationship between SF3B1 abnormalities and tumorigenesis, studies onthe pleiotropic splicing abnormalities are necessary.

Among these types of abnormal splicing regulation, it was reported that diverse SF3B1 mutationsconverge on the repression of BRD9, which is a core component of the recently described noncanonicalBAF chromatin-remodeling complex, which plays a suppressor role in tumorigenesis [62]. That studyfound that mutant SF3B1 recognizes an aberrant intronic BP within BRD9, thereby inducing theinclusion of a poison exon. This results in the repression of BRD9 mediated by NMD, thus promotingtumor growth and metastasis. These results indicate that the poison exon of BRD9 becomes a targetof therapeutic potential in SF3B1-mutated cancers. Indeed, tumor-suppressive effects of correctingBRD9 mis-splicing with multiple methods, including antisense oligonucleotides, were achieved [62].In general, it is thought that the multiple splicing alterations may cooperatively contribute to thepathogenesis of cancers. Thus, recent studies suggest the potential of new therapeutics targetingmis-spliced transcripts in anticancer treatment (Figure 3B).


discovered to be cytotoxic compounds to cancer cells in early research. Based on this information, a splicing inhibitor is expected to act as an anti-cancer drug with a new mechanistic movement (Figure 3A). The first splicing inhibitor to enter clinical trials was pladienolide derivative E7107 [179,180]. Unfortunately, this trial was discontinued because of vision loss occurring in some study participants [180]. Recently, H3B-8800 showed greater preferential cytotoxicity in spliceosome-mutant cells than E7107 by retaining short and GC-rich introns, which are enriched in genes encoding spliceosome components [157]. The enrichment of retained introns in mRNAs encoding spliceosome factors provides a rationale for H3B-8800 giving a preferential killing effect to spliceosome-mutant cancer cells compared with normal cells with a wild-type spliceosome. Consequently, H3B-8800 selectively killed acute myeloid leukemia cells and xenograft tumors. H3B-8800 entered phase 1 clinical trials [157], and it is expected to be the first anti-cancer drug with splicing inhibition (Figure 3B).

Figure 3. Availability of compounds with splicing inhibition activities. (A) Effects of splicing inhibitors on normal cells, cancer cells, and cancer cells with mutations in splicing factors. Cancer cells have more transcripts than normal cells, resulting in splicing stress. In addition, cells with mutations in the splicing factor cause splicing abnormalities. These splicing factor mutations are heterozygous, suggesting that wild-type splicing factors have an essential role in cell survival in these cancers. Cancer cells with or without splicing factor mutations are, thus, more sensitive to splicing inhibition than normal cells. (B) The anti-cancer effect of various splicing inhibitions. SF3B1 inhibitors, such as H3B-8800, are anticipated for clinical use. Food-derived compounds with similar splicing inhibition activity were also reported to have anti-cancer effects, and they are expected to have cancer-preventing effects. In addition, inhibition of mis-splicing on BRG9, which is widely observed in cancers with an SF3B1 mutation, using antisense oligonucleotides (ASO) is expected to be a new strategy for cancer treatment.

4.2. Compounds Regulating mRNA Splicing by G-Quadruplex Control

Figure 3. Availability of compounds with splicing inhibition activities. (A) Effects of splicing inhibitorson normal cells, cancer cells, and cancer cells with mutations in splicing factors. Cancer cells have moretranscripts than normal cells, resulting in splicing stress. In addition, cells with mutations in the splicingfactor cause splicing abnormalities. These splicing factor mutations are heterozygous, suggesting thatwild-type splicing factors have an essential role in cell survival in these cancers. Cancer cells with orwithout splicing factor mutations are, thus, more sensitive to splicing inhibition than normal cells. (B)The anti-cancer effect of various splicing inhibitions. SF3B1 inhibitors, such as H3B-8800, are anticipatedfor clinical use. Food-derived compounds with similar splicing inhibition activity were also reported tohave anti-cancer effects, and they are expected to have cancer-preventing effects. In addition, inhibitionof mis-splicing on BRG9, which is widely observed in cancers with an SF3B1 mutation, using antisenseoligonucleotides (ASO) is expected to be a new strategy for cancer treatment.

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3.1.2. SRSF2

SRSF2 is a member of the SR protein family that promotes exon inclusion by binding to theESE sequences, CCNG or GGNG, through its RNA recognition motif (RRM) domain [9]. SRSF2mutations consistently affect the P95 residue and have increased binding affinity toward CCNG, buthave decreased binding affinity toward the GGNG sites [107–109]. As a result, global alterations ofsplicing, including exon inclusion and exclusion, are induced by SRSF2 mutation [63,107–109]. SRSF2mutants promote the splicing alteration of key hematopoietic regulators, such as enhancer of zestehomolog 2 (EZH2), which impairs hematopoietic differentiation. Because SRSF2 mutation did not affectprotein–protein interactions with key splicing factors in one study [107], it was assumed that SRSF2mutations affect the conformation of the RNA-binding domain [108,110]. The structural information ofSRSF2 will uncover the mechanism of abnormal splicing induced by SRSF2 mutation.

In addition to the splicing alteration in SRSF2 mutant cells, DNA damage is also induced [111].SRSF2 mutation causes impairment of transcription pause release and induces R-loop formation.Accumulation of R-loops often results in increased cellular stress that leads to genomic instability [112].The chromosomal instability could be a major driving force in tumorigenesis and cancer evolution.

3.1.3. U2AF1

Recurrent hotspot mutations in U2AF1 at the Zn-finger domain (S34 or Q157) werereported [113,114]. These mutations altered the RNA-binding specificity [114–117] and the splicingkinetics [118], resulting in a wide variety of splicing outcomes. Changes in the cassette exon wereobserved most frequently in these mutations [94,115,119–123]. Although it was reported that theU2AF1 mutant also induces abnormal splicing of EZH2 [63]; other splicing alterations are not linkedto a phenotype, leading some investigators to propose a nuclear RNA that processes defects such asalterations in 3′UTRs [124] or an increase in R-loops induced by the U2AF1 mutant [111,125].

3.1.4. U1 snRNP

A recurrent A-to-C and A-to-G mutation in the third base of the U1 snRNA occurs at part of ahighly conserved 5′ ss recognition sequence (nucleotides 3–10) of U1, which forms base pairs directlywith the 5′ ss [97,126]. This mutation changed the preferential A–U base-pairing between the U1snRNA and the 5′ ss to C–G base-pairing, and it caused alternative 5′ cryptic splicing and alterationof the splicing pattern in multiple genes, including known drivers and repressors of cancer [97,98].These events are thought to be specific to U1 snRNA mutations because SF3B1 mutations tend not toshare these abnormal types of splicing [97].

As a novel link between cancer progression and splicing factors through noncanonical roles, theassociation with splicing factor U2AF1 and translational regulation was reported [127]. That studyrevealed that U2AF1 and U2AF2, bound to the 5′ UTR, were located on hundreds of mature RNA in thecytoplasm and functioned as a translational repressor. The recurrent cancer-associated hotspot mutation(S34F) in U2AF1 caused loss of binding and translational de-repression, resulting in increased synthesisof the IL8 chemokine, which contributes to metastasis, inflammation, and cancer progression [128,129].In addition to the contribution of U2AF1, it was reported that SF3B4, a component of the SF3B complex,functions as a cofactor for p180, an essential factor for high-rate protein synthesis on the ER, and itplays a key role in enhanced translation [130]. These findings suggest that mRNA splicing factors havemultiple roles in translation, and cancer-related mutations of mRNA splicing factors affect translation,as well as splicing.

3.2. Spliceosome Abnormality and Neurodegenerative Disorders

It is widely accepted concept that abnormalities of a number of RBPs are related to neurologicaldiseases. Aberrant activity and localization of two RBPs, TDP-43 and FUS RNA binding protein (FUS),are implicated as the pathogenicity of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia

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(FTD), two fatal neurodegenerative disorders that share clinical, genetic, and pathologic hallmarks [131].These proteins were found to mediate a number of pathways related to RNA metabolism in thecytoplasm. In each disease, TDP-43 and FUS form mRNP aggregates in the cytoplasm [132–134].The pathological consequences of mRNP aggregates are believed to be multifactorial in nature, but theirroles are not yet completely defined. These aggregates disturb their normal functions by disruptingsplicing [1].

Several reports demonstrate that spliceosomal components in the nucleus are mislocalized into anaggregate commonly observed in these neurodegenerative disorders [135–137]. The U1 snRNP, themost abundant FUS interacting complex, co-mislocalizes with FUS to the cytoplasm [135]. In addition,cytoplasmic aggregation of TDP-43 causes mislocalization of spliceosome components [136]. Inzebrafish, knockdown of U1 snRNP components caused the truncation of the motor neuron axon,and similar phenotypes were observed by knockdown of FUS and survival motor neuron protein(SMN) [135]. Alzheimer’s disease (AD) is an age-related neurodegenerative disorder characterized bysynaptic dysfunction, amyloid plaques, and neurofibrillary tangles formed by the aggregation of Tauprotein encoded by the microtubule associated protein tau (MAPT) gene [138]. In the brains of patients withAD, increased aggregation of insoluble U1 snRNP was identified by proteomic analysis [137]. Previousstudies showed that the U1 snRNP co-aggregates with Tau in the neurofibrillary tangles in humanAD postmortem brain tissue [139,140]. Similar findings were reported using in vitro experiments andMAPT transgenic mice [141–143]. In Drosophila, panneuronal Tau expression triggers aggregation ofU1-specific spliceosomal proteins [144]. That study suggested that mislocalization of spliceosomalcomponents is also associated with these neurodegenerative disorders.

The functions of TDP-43 include repressing the splicing of nonconserved cryptic exons, maintainingintron integrity and preventing cell death [56,145]. RNA-seq analysis in human postmortem brainwith TDP-43 mutations revealed cryptic exon expression [56]. This feature is common in ALS/FTDpatients [56,138]. Whether the mislocalization of spliceosomal components induced by the aggregationof FUS or TDP-43 directly contributes to the cryptic splicing is still unknown. However, in AD, Taupathology associates with splicing errors, including cryptic splicing and intron retention in humanbrains [144]. The mislocalization of the U1 snRNP-mediated aggregation of Tau causes a loss of nuclearU1 snRNP. A similar profile of cryptic splicing and intron retention was reported in Tau transgenicfly and small nuclear ribonucleoprotein-associated protein B (SmB), a component of snRNP, mutantfly [144,146]. Furthermore, mutation of SmB causes progressive neurodegeneration. The loss of theU2 snRNA also induced cryptic splice junctions and intron retention along with prominent cerebellardegeneration. These reports suggested that disruption or alteration of spliceosome function causessevere and toxic transcriptome formation and neurodegenerative diseases. In addition to the aboveevidence, findings showed that, preceding the onset of neurodegeneration, splicing changes weredetectable in young flies [147]. Furthermore, direct manipulation of a core spliceosome componentalso caused neurodegeneration, which raises the possibility that splicing errors are likely a causerather than a consequence of neurodegenerative disorders. The observation that alternative splicingoccurred at the highest rate in the brain is consistent with the idea that the regulation of splicing in thebrain has an important role in neuronal diversity and brain health [148]. Investigating connectionsbetween the unique role of core spliceosomal components and a molecular mechanism associatedwith neurodegenerative disorders will increase the understanding of therapeutic strategies targetingthese factors.

4. Compounds that Control mRNA Splicing

As mentioned above, mRNA splicing is a complicated and dynamic process with a number ofinteracting splicing factors. One effective investigation strategy is to analyze the mechanism of mRNAprocessing using chemical compounds as biological tools that function to inhibit mRNA processes.In this section, we discuss the various compounds with splicing inhibitors discovered recently andmodulators of rG4 formation.

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4.1. Chemical Compounds That Control mRNA Splicing

Various compounds with inhibitory activity on splicing were identified from severalmicroorganisms. Some of these compounds were found as cancer-specific effectors in early research,and the studies that followed revealed that the target proteins of these compounds were mRNA splicingfactors. Synthetic derivatives of these compounds are, therefore, established as more effective splicinginhibitors. To date, the best-studied group of splicing inhibitors involves SF3B1 inhibitors, whichcontain pladienolide B (PlaB), spliceostatin A (SSA), GEX1A, and their analogues. We briefly introducerepresentative SF3B1 inhibitors and isoginkgetin, which inhibits a different target (Table 1).

4.1.1. Representative SF3B1 Inhibitors and Isoginkgetin

SF3B1 inhibitors were individually identified as having distinct structures based on differentassays. PlaB, isolated from a natural product derived from Streptomyces platensis Mer-11107, has cellularsplicing inhibition activity [149]. E7107 has enhanced stability compared with PlaB and directly bindsto SF3B3, a component of the SF3B complex. This results in the inhibition of SF3B1 and the impairmentof U2 snRNP interaction with pre-mRNA [149]. Other studies reported that E7107 interacts with theSF3B complex in the branch point adenosine-binding pocket [150,151], which results in reducing thestability of early “A complex” formation by weakening the interaction between the U2 snRNA andpre-mRNA [152–156]. H3B-8800, an analogue of PlaB, was identified as a compound that competedwith PlaB for binding to SF3B complexes. H3B-8800 received focus as a next-generation splicinginhibitor to enter clinical trials because of its similar, but not identical, activity to E7107, as describedlater [157].

SSA is a methylated derivative of the natural product, FR901464, which was isolated from thefermented broth of the bacterium Pseudomonas sp. as an anticancer compound [158]. In subsequentresearch, it was found that SSA binds to the SF3B complex, decreases the U2 snRNA interactionswith the BP and results in the inhibition of splicing [153]. Sudemycin was designed based on thepharmacophore model between FR901464 and PlaB. Sudemycin shows splicing inhibition activity in asimilar manner to SSA, and exhibits better chemical stability and half-maximal inhibitory concentration(IC50) values in cell lines [159,160].

GEX1A was originally isolated from a culture broth of Streptomyces sp. [161] and serves as asplicing inhibitor that specifically impairs the SF3B function by binding to SF3B1 [162]. Studies involvedin the search for synthetic analogues of GEX1A revealed that the splicing inhibitory potency of theanalogues and the modification of carboxylic acid moiety are well correlated with the antiproliferativeactivity against cancer cell lines [163,164].

The plant-derived splicing inhibitor, isoginkgetin, was identified from the leaves of the gingkotree by the screening of its mRNA splicing inhibitor [165]. Biflavones such as isoginkgetin belongto a subclass of the plant flavonoid family, which were reported to show anti-cancer activity [166].Isoginkgetin inhibits splicing by preventing the stable recruitment of the U4/U5/U6 tri-small nuclearribonucleoprotein, resulting in the accumulation of prespliceosomal A complex [165].

4.1.2. Molecular Mechanism of Splicing Investigated by Splicing Inhibitors

Inhibitors introduced in this review were also used to provide detailed mechanistic insight intospliceosome formation and remodeling, as well as its impact on gene expression in cells.

SF3B1 inhibitors are powerful tools to elucidate the function of SF3B1 and the SF3B complex incomplicated splicing mechanisms such as the spliceosome assembly. Previously, the association ofSF3B1 with the recruitment and/or stabilization of the U2 snRNP at the BP was reported [100,167–170].However, the mechanisms of BP recognition by the U2 snRNP and SF3B1 are still unknown. The study,using the chemical compound, E7107, which affects U2 snRNP interactions at the BP, concluded thatSF3B1 plays a role in mediating a conformational change of the U2 snRNP [154]. Similarly, SSAreduces the fidelity of U2 snRNA interactions with the BP, suggesting that SF3B1 participates in BP

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discrimination [153]. Cryo-EM structure analysis of the SF3B complex with or without E7107 showedthat the compound bound to the mRNA-unbound SF3B complex and this SF3B complex was differentfrom the RNA-bound closed conformation. This provided evidence that RNA and possibly othersplicing factors trigger a conformational change in the SF3B complex for the spliceosome assembly,and E7107 causes splicing abnormalities by disrupting the conformational change [171]. In addition,using SSA and PlaB, it was reported that SF3B1 is involved not only in the early splicing reactionby BP recognition but also in the exon-ligation reaction [172], suggesting additional roles for SF3B1throughout the splicing process.

Table 1. Chemical compounds which control splicing.

Name of Compounds and Chemical Formulas Origin and Reference Features of Inhibitor

pladienolide B (PlaB)


Inhibitors introduced in this review were also used to provide detailed mechanistic insight into spliceosome formation and remodeling, as well as its impact on gene expression in cells. SF3B1 inhibitors are powerful tools to elucidate the function of SF3B1 and the SF3B complex in complicated splicing mechanisms such as the spliceosome assembly. Previously, the association of SF3B1 with the recruitment and/or stabilization of the U2 snRNP at the BP was reported [100,167–170]. However, the mechanisms of BP recognition by the U2 snRNP and SF3B1 are still unknown. The study, using the chemical compound, E7107, which affects U2 snRNP interactions at the BP, concluded that SF3B1 plays a role in mediating a conformational change of the U2 snRNP [154]. Similarly, SSA reduces the fidelity of U2 snRNA interactions with the BP, suggesting that SF3B1 participates in BP discrimination [153]. Cryo-EM structure analysis of the SF3B complex with or without E7107 showed that the compound bound to the mRNA-unbound SF3B complex and this SF3B complex was different from the RNA-bound closed conformation. This provided evidence that RNA and possibly other splicing factors trigger a conformational change in the SF3B complex for the spliceosome assembly, and E7107 causes splicing abnormalities by disrupting the conformational change [171]. In addition, using SSA and PlaB, it was reported that SF3B1 is involved not only in the early splicing reaction by BP recognition but also in the exon-ligation reaction [172], suggesting additional roles for SF3B1 throughout the splicing process.



pladienolide B

(PlaB)

be derived from

Streptomyces platensis

Mer-11107 [149]

inhibit splicing

E7107

an analog of PlaB

[149–156]

directly bind to SF3B complex

and inhibit SF3B to interact

with pre-mRNA

H3B-8800

an analog of PlaB [157]

have entered clinical trials as

anti-cancer drug because of

preferential killing effect to

spliceosome-mutant cancer

FR901464

be derived from

fermented broth of

bacterium Pseudomonas sp

[158]

inhibit splicing

be derived from Streptomycesplatensis Mer-11107 [149] inhibit splicing

E7107





pladienolide B

(PlaB)

be derived from


Mer-11107 [149]

inhibit splicing

E7107

an analog of PlaB

[149–156]



with pre-mRNA

H3B-8800






FR901464

be derived from

fermented broth of


[158]

inhibit splicing

an analog of PlaB [149–156] directly bind to SF3B complex and inhibitSF3B to interact with pre-mRNA

H3B-8800





pladienolide B

(PlaB)

be derived from


Mer-11107 [149]

inhibit splicing

E7107

an analog of PlaB

[149–156]



with pre-mRNA

H3B-8800






FR901464

be derived from

fermented broth of


[158]

inhibit splicing

an analog of PlaB [157]have entered clinical trials as anti-cancer

drug because of preferential killing effect tospliceosome-mutant cancer

FR901464





pladienolide B

(PlaB)

be derived from


Mer-11107 [149]

inhibit splicing

E7107

an analog of PlaB

[149–156]



with pre-mRNA

H3B-8800






FR901464

be derived from

fermented broth of


[158]

inhibit splicing be derived from fermented broth

of bacterium Pseudomonas sp. [158] inhibit splicing

spliceostatin A (SSA)


spliceostatin A

(SSA)

an analog of FR901464

[153]



with pre-mRNA

sudemycin C1

be designed based on

pharmacophore model

between FR901464 and

PlaB [159,160]

exhibit better chemical

stability

than SSA and PlaB

GEX1A

be derived from

a culture broth of

Streptomyces sp.

[161,162]



with pre-mRNA

9g, synthetic analogue of GEX1A

an analog of GEX1A

[163,164]

be expected as the lead

compound for the

development of novel

antitumor agents

Isoginkgetin

be derived from the

leaves of the gingko tree

[165,166]

prevent transition of the

spliceosome

an analog of FR901464 [153] directly bind to SF3B complex and inhibitSF3B to interact with pre-mRNA

sudemycin C1


spliceostatin A

(SSA)


[153]



with pre-mRNA

sudemycin C1


pharmacophore model


PlaB [159,160]


stability

than SSA and PlaB

GEX1A

be derived from

a culture broth of

Streptomyces sp.

[161,162]



with pre-mRNA


an analog of GEX1A

[163,164]


compound for the


antitumor agents

Isoginkgetin

be derived from the


[165,166]


spliceosome

be designed based onpharmacophore model between

FR901464 and PlaB [159,160]

exhibit better chemical stability than SSAand PlaB

GEX1A


spliceostatin A

(SSA)


[153]



with pre-mRNA

sudemycin C1


pharmacophore model


PlaB [159,160]


stability

than SSA and PlaB

GEX1A

be derived from

a culture broth of

Streptomyces sp.

[161,162]



with pre-mRNA


an analog of GEX1A

[163,164]


compound for the


antitumor agents

Isoginkgetin

be derived from the


[165,166]


spliceosome

be derived from a culture broth ofStreptomyces sp. [161,162]

directly bind to SF3B complex and inhibitSF3B to interact with pre-mRNA



spliceostatin A

(SSA)


[153]



with pre-mRNA

sudemycin C1


pharmacophore model


PlaB [159,160]


stability

than SSA and PlaB

GEX1A

be derived from

a culture broth of

Streptomyces sp.

[161,162]



with pre-mRNA


an analog of GEX1A

[163,164]


compound for the


antitumor agents

Isoginkgetin

be derived from the


[165,166]


spliceosome

an analog of GEX1A [163,164] be expected as the lead compound for thedevelopment of novel antitumor agents

isoginkgetin


spliceostatin A

(SSA)


[153]



with pre-mRNA

sudemycin C1


pharmacophore model


PlaB [159,160]


stability

than SSA and PlaB

GEX1A

be derived from

a culture broth of

Streptomyces sp.

[161,162]



with pre-mRNA


an analog of GEX1A

[163,164]


compound for the


antitumor agents

Isoginkgetin

be derived from the


[165,166]


spliceosome

be derived from the leaves of thegingko tree [165,166] prevent transition of the spliceosome

hinokiflavone


Hinokiflavone

an analog of isoginkgetin

[173]

induce SUMOylation of

splicing factors by inhibiting

SENP1 activity, and prevent

transition of the spliceosome

The relationship between splicing factor and SUMOylation, a post-translational modification, was partially clarified by hinokiflavone, which is an analogue of isoginkgetin [173]. Previously, spliceosomal proteins were revealed as SUMO conjugation targets; however, little is known about the involvement of SUMO in spliceosome biogenesis and splicing regulation [174]. Hinokiflavone induced SUMOylation of pre-mRNA splicing factors, which contain six components of the U2 snRNP spliceosome subunit, by inhibiting SUMO protease SUMO-specific peptidase 1 (SENP1) activity. Consequently, hinokiflavone prevented transition of the spliceosome from its A to B complexes, resulting in global splicing modulation. It was also reported that inhibition of pre-mRNA processing factor 3 (PRPF3) SUMOylation prevented the interaction of U4/U6 di-snRNP with U5 to form tri-snRNP [175]. These reports suggested the notion that SUMOylation cycles were involved in spliceosome assembly and catalytic activity, and that they also affected alternative splicing regulation. An investigation using a compound like hinokiflavone is expected to facilitate the relationship between SUMO modification and alternative splicing.

These splicing-modulating compounds generally function as splicing inhibitors. However, affected transcripts are part of mRNAs. The sequence features which caused aberrant splicing by SSA and sudemycin, E7107, and H3B-8800 treatment were analyzed from recent transcriptome analyses [156,157,176]. Commonly, compound-induced retained introns are typically shorter and display a higher GC content and weaker polypyrimidine tracts and BP sequence. In addition, the presence of multiple BPs has an important role in determining sensitivity [156]. Interestingly, despite their structural similarities, SSA and sudemycin show common and differential effects on splicing regulation. SSA generally displays stronger effects on intron retention, and sudemycin affects exon skipping [156]. H3B-8800 is also more effective than E7107 on short introns that are rich in GC content [157]. Understanding of the relationships between these compounds and the differences in genome-wide splicing control will provide support for high-resolution observations of the splicing mechanism.

4.1.3. Focusing on Splicing Regulation for Therapeutics of Cancer

Mutations of splicing factors found in various cancers are all heterozygous, probably because wild-type splicing factor has an essential role for cell survival in these cancers. Cancers that have the splicing factor mutation, therefore, show higher sensitivity to splicing inhibitors [177]. Some cancers without the splicing factor mutation also show sensitivity to splicing inhibitors. This observation is explained by the stress in splicing because primary transcripts are more abundantly expressed in cancer cells than in normal cells [178]. In fact, as previously mentioned, many splicing inhibitors were discovered to be cytotoxic compounds to cancer cells in early research. Based on this information, a splicing inhibitor is expected to act as an anti-cancer drug with a new mechanistic movement (Figure 3A). The first splicing inhibitor to enter clinical trials was pladienolide derivative E7107 [179,180]. Unfortunately, this trial was discontinued because of vision loss occurring in some study participants [180]. Recently, H3B-8800 showed greater preferential cytotoxicity in spliceosome-mutant cells than E7107 by retaining short and GC-rich introns, which are enriched in genes encoding spliceosome components [157]. The enrichment of retained introns in mRNAs encoding spliceosome factors provides a rationale for H3B-8800 giving a preferential killing effect to spliceosome-mutant cancer

an analog of isoginkgetin [173]induce SUMOylation of splicing factors by

inhibiting SENP1 activity, and preventtransition of the spliceosome

The relationship between splicing factor and SUMOylation, a post-translational modification,was partially clarified by hinokiflavone, which is an analogue of isoginkgetin [173]. Previously,spliceosomal proteins were revealed as SUMO conjugation targets; however, little is known about

Int. J. Mol. Sci. 2020, 21, 2026 14 of 27

the involvement of SUMO in spliceosome biogenesis and splicing regulation [174]. Hinokiflavoneinduced SUMOylation of pre-mRNA splicing factors, which contain six components of the U2snRNP spliceosome subunit, by inhibiting SUMO protease SUMO-specific peptidase 1 (SENP1) activity.Consequently, hinokiflavone prevented transition of the spliceosome from its A to B complexes, resultingin global splicing modulation. It was also reported that inhibition of pre-mRNA processing factor 3(PRPF3) SUMOylation prevented the interaction of U4/U6 di-snRNP with U5 to form tri-snRNP [175].These reports suggested the notion that SUMOylation cycles were involved in spliceosome assemblyand catalytic activity, and that they also affected alternative splicing regulation. An investigation usinga compound like hinokiflavone is expected to facilitate the relationship between SUMO modificationand alternative splicing.

These splicing-modulating compounds generally function as splicing inhibitors. However,affected transcripts are part of mRNAs. The sequence features which caused aberrant splicingby SSA and sudemycin, E7107, and H3B-8800 treatment were analyzed from recent transcriptomeanalyses [156,157,176]. Commonly, compound-induced retained introns are typically shorter anddisplay a higher GC content and weaker polypyrimidine tracts and BP sequence. In addition,the presence of multiple BPs has an important role in determining sensitivity [156]. Interestingly,despite their structural similarities, SSA and sudemycin show common and differential effects onsplicing regulation. SSA generally displays stronger effects on intron retention, and sudemycinaffects exon skipping [156]. H3B-8800 is also more effective than E7107 on short introns that arerich in GC content [157]. Understanding of the relationships between these compounds and thedifferences in genome-wide splicing control will provide support for high-resolution observations ofthe splicing mechanism.

4.1.3. Focusing on Splicing Regulation for Therapeutics of Cancer

Mutations of splicing factors found in various cancers are all heterozygous, probably becausewild-type splicing factor has an essential role for cell survival in these cancers. Cancers that have thesplicing factor mutation, therefore, show higher sensitivity to splicing inhibitors [177]. Some cancerswithout the splicing factor mutation also show sensitivity to splicing inhibitors. This observationis explained by the stress in splicing because primary transcripts are more abundantly expressed incancer cells than in normal cells [178]. In fact, as previously mentioned, many splicing inhibitors werediscovered to be cytotoxic compounds to cancer cells in early research. Based on this information,a splicing inhibitor is expected to act as an anti-cancer drug with a new mechanistic movement(Figure 3A).

The first splicing inhibitor to enter clinical trials was pladienolide derivative E7107 [179,180].Unfortunately, this trial was discontinued because of vision loss occurring in some studyparticipants [180]. Recently, H3B-8800 showed greater preferential cytotoxicity in spliceosome-mutantcells than E7107 by retaining short and GC-rich introns, which are enriched in genes encodingspliceosome components [157]. The enrichment of retained introns in mRNAs encoding spliceosomefactors provides a rationale for H3B-8800 giving a preferential killing effect to spliceosome-mutantcancer cells compared with normal cells with a wild-type spliceosome. Consequently, H3B-8800selectively killed acute myeloid leukemia cells and xenograft tumors. H3B-8800 entered phase 1 clinicaltrials [157], and it is expected to be the first anti-cancer drug with splicing inhibition (Figure 3B).

4.2. Compounds Regulating mRNA Splicing by G-Quadruplex Control

Initially, compounds with regulating rG4 structures associated with translational control wereexplored. RGB-1, RR82, and RR110, which bind selectively to rG4, affect the stability of NRASmRNA rG4 and the translational efficiency of the NRAS 5′ UTR (Table 2) [181,182]. TRF2 is a proteinwith a central role in telomere maintenance. Three bisquinolinium compounds, 360A, PhenDC3,and PhenDC6, potentially bind to the TRF2 mRNA rG4 to alter its translation [183]. In addition,a compound that affected alternative mRNA splicing through regulating rG4 structures was also

Int. J. Mol. Sci. 2020, 21, 2026 15 of 27

found. Previously, it was shown that there are two rG4 forming sites in Bcl-X mRNA, each beinglocated close to an individual alternative 5′ ss. The compound, GQC-05, affects only one site byenhancing the stability of rG4 dependent on its structure [184]. The binding results in the reducedusage of the major 5′ ss that expresses the anti-apoptotic isoform of Bcl-X and the increased usage ofan alternative 5′ ss that expresses a pro-apoptotic isoform. Moreover, alteration of splicing inducesapoptosis. It was also reported that emetine and its analogue, cephaeline, disrupted rG4, resulting inthe inhibition of rG4-dependent alternative splicing. Transcriptome analysis revealed that emetineglobally regulates alternative splicing with variable exons that contain rG4 near proximal splice sites.This analysis revealed that rG4 controls alternative splicing at a genome-wide scale. Interestingly,emetine promotes the EMT state, suggesting that small molecules may alter cell fates associated withcancer progression [72].

Table 2. Modulators of G-quadroplex which control translation or splicing.


RGB-1


5′ ss that expresses a pro-apoptotic isoform. Moreover, alteration of splicing induces apoptosis. It was also reported that emetine and its analogue, cephaeline, disrupted rG4, resulting in the inhibition of rG4-dependent alternative splicing. Transcriptome analysis revealed that emetine globally regulates alternative splicing with variable exons that contain rG4 near proximal splice sites. This analysis revealed that rG4 controls alternative splicing at a genome-wide scale. Interestingly, emetine promotes the EMT state, suggesting that small molecules may alter cell fates associated with cancer progression [72].



RGB-1

synthetic molecules

[181,182]

modulate translational

efficiency of TRF2

RR82

synthetic molecules

[181,182]


efficiency of TRF2

RR110

an analog of RR82

[181,182]


efficiency of TRF2

360A

synthetic molecules

[183]


efficiency of TRF2

synthetic molecules [181,182] modulate translational efficiency of TRF2

RR82





RGB-1

synthetic molecules

[181,182]


efficiency of TRF2

RR82

synthetic molecules

[181,182]


efficiency of TRF2

RR110

an analog of RR82

[181,182]


efficiency of TRF2

360A

synthetic molecules

[183]


efficiency of TRF2

synthetic molecules [181,182] modulate translational efficiency of TRF2

RR110





RGB-1

synthetic molecules

[181,182]


efficiency of TRF2

RR82

synthetic molecules

[181,182]


efficiency of TRF2

RR110

an analog of RR82

[181,182]


efficiency of TRF2

360A

synthetic molecules

[183]


efficiency of TRF2

an analog of RR82 [181,182] modulate translational efficiency of TRF2

360A





RGB-1

synthetic molecules

[181,182]


efficiency of TRF2

RR82

synthetic molecules

[181,182]


efficiency of TRF2

RR110

an analog of RR82

[181,182]


efficiency of TRF2

360A

synthetic molecules

[183]


efficiency of TRF2

synthetic molecules [183] modulate translational efficiency of TRF2

PhenDC3


PhenDC3

synthetic molecules

[183]


efficiency of TRF2

PhenDC6

an analog of PhenDC3

[183]


efficiency of TRF2

GCQ-05

synthetic molecules

[184]

modulate alternative splicing

of Bcl-X

Emetine

be contained in ipecas

root [72]


with variable exons which

contain rG4 near proximal

splice sites

Cephaeline

an analog of emetine [72] show similar effect with

emetine

Regulation of mRNA processing by rG4 is still unclear. Newly identified compounds that regulate rG4 will uncover the underlying RNA processing mechanism by rG4. In addition, several studies also indicated that rG4 structures are associated with human diseases, including neurological disorders [99] and cancer [185,186]. Uncovering these relationships will aid in therapy against diseases caused by rG4.

4.3. Food-Derived Natural Compounds Capable of Controlling mRNA Splicing

synthetic molecules [183] modulate translational efficiency of TRF2

PhenDC6


PhenDC3

synthetic molecules

[183]


efficiency of TRF2

PhenDC6


[183]


efficiency of TRF2

GCQ-05

synthetic molecules

[184]


of Bcl-X

Emetine


root [72]




splice sites

Cephaeline


emetine



an analog of PhenDC3 [183] modulate translational efficiency of TRF2

GCQ-05


PhenDC3

synthetic molecules

[183]


efficiency of TRF2

PhenDC6


[183]


efficiency of TRF2

GCQ-05

synthetic molecules

[184]


of Bcl-X

Emetine


root [72]




splice sites

Cephaeline


emetine



synthetic molecules [184] modulate alternative splicing of Bcl-X

emetine


PhenDC3

synthetic molecules

[183]


efficiency of TRF2

PhenDC6


[183]


efficiency of TRF2

GCQ-05

synthetic molecules

[184]


of Bcl-X

Emetine


root [72]




splice sites

Cephaeline


emetine



be contained in ipecas root [72]modulate alternative splicing with variable

exons which contain rG4 near proximalsplice sites

cephaeline


PhenDC3

synthetic molecules

[183]


efficiency of TRF2

PhenDC6


[183]


efficiency of TRF2

GCQ-05

synthetic molecules

[184]


of Bcl-X

Emetine


root [72]




splice sites

Cephaeline


emetine



an analog of emetine [72] show similar effect with emetine

Int. J. Mol. Sci. 2020, 21, 2026 16 of 27

Regulation of mRNA processing by rG4 is still unclear. Newly identified compounds that regulaterG4 will uncover the underlying RNA processing mechanism by rG4. In addition, several studies alsoindicated that rG4 structures are associated with human diseases, including neurological disorders [99]and cancer [185,186]. Uncovering these relationships will aid in therapy against diseases caused by rG4.


The above compounds are anticipated for clinical use. In addition, food-derived naturalcompounds with similar activity to the above compounds may function as anti-cancer drugs. Recentstudies suggested that compounds originating from food also contain various anti-cancer activities,showing a variety of physiological activities within cells and the whole body (Figure 3B). For example,daily intake of bioactive food compounds is expected to be effective for preventing chronic diseases.Polyphenols and carotenoids have antioxidant activity and function to prevent lifestyle-relateddiseases [187–189]. In this section, we discuss some reports that were published on food-derivednatural compounds with the ability to modulate mRNA processing.

Resveratrol is a polyphenolic flavonoid found in grape skins, grape seeds, and red wine [190],and it was shown to protect against cardiovascular disease, type 2 diabetes, and neurological disorders(Table 3) [190–192]. It was found that resveratrol modulates alternative splicing of SRSF3 and SMN2mRNAs. The splicing abnormalities of these mRNAs are associated with the above-mentioneddiseases [193]. This effect may be partly due to the ability of resveratrol to affect the protein levelof several RNA processing factors, ASF/SF2, hnRNP A1, and human antigen R (HuR). Previously,although the SIRT1 protein was regarded as a major target of resveratrol, the knockdown of SIRT1did not modulate alternative splicing of SRSF3 and SMN2 mRNAs, suggesting that another splicingregulatory protein is regulated by resveratrol for modulating these mRNAs.

Caffeine, abundant in coffee and tea, modulates the cell cycle and growth arrest, and inducesapoptosis via the expression of various alternatively spliced p53 isoforms [194]. In one study, caffeinealtered the expression levels of the p53α and p53β isoforms that are mediated by the downregulationof the SRSF3 mRNA and protein. In addition to p53 isoforms, other SRSF3 target genes were alsoalternatively spliced in response to caffeine treatment [195]. That study provided a new pathway ofcaffeine-modulated tumor suppression via the alternative splicing of target genes of SRSF3.

Curcumin is a dietary polyphenolic compound enriched in the roots of turmeric with a broadtherapeutic potential for cancer because of curcumin’s antitumor activity in various cancer cells [196,197].Recently, it was reported that curcumin caused a splicing switch from a cancer-specific PKM2 isoformto normal PKM1 isoform in head and neck cancer cells, resulting in reduced tumor growth. In addition,global transcriptome analysis of curcumin-treated cells revealed curcumin’s effect on the alternativesplicing of various genes involved in head and neck cancer [198].

We examined whether food-derived compounds provide inhibitory activity toward mRNAprocessing in the nucleus using a previously established screening system [199,200]. We identifiedthe activity of inhibiting mRNA processing in the soybean-derived isoflavonoid fraction [200,201].Furthermore, this activity was mainly exerted by compounds with a flavone skeleton [200]. Amongthem, apigenin and luteolin exhibited the strongest activity among 21 compounds with a flavoneskeleton [202]. These compounds most intensely interacted with the U2 and U5 snRNP, suggestingthat apigenin and luteolin were associated with spliceosomal components to directly prevent thefunction of spliceosomes, thus affecting alternative splicing at the genome-wide level. We observedmore prominent sensitivity of tumorigenic cells to apigenin and luteolin than that observed fornontumorigenic cells, suggesting the potential anti-cancer activity of these compounds. We alsoscreened for active constituents from spices and detected the inhibition of mRNA processing activityin ginger, cinnamon, and clove extracts. It seems that there may be many constituents with inhibitoryactivity toward mRNA processing in spices. Furthermore, 6-gingerol and 6-shogaol, active componentsin ginger, showed inhibitory activity toward mRNA processing [203]. The anti-cancer activity that

Int. J. Mol. Sci. 2020, 21, 2026 17 of 27

was observed in ginger extract and 6-gingerol [204] might be partly exerted through anti-mRNAprocessing activity.

Table 3. Food derived compounds which control mRNA processing or splicing.


Resveratrol


Resveratrol

be contained in grape

skins, grape seeds and

red wine [191–194]


of SRSF3 and SMN2

Caffeine

be contained in coffee

and tea [195,196]


of p53, and SRSF3 target

mRNAs

Curcumin

be contained in the roots

of turmeric [197-199]


of PKM2 and various mRNAs

involved in head and neck

cancer

Apigenin

be contained in parsley

and celery [203]

interact with the U2 and U5

snRNP and affect alternative

splicing

Luteolin


and celery [203]

show similar effect with

apigenin

be contained in grape skins, grape seedsand red wine [191–194]

modulate alternative splicing of SRSF3 andSMN2

Caffeine


Resveratrol



red wine [191–194]


of SRSF3 and SMN2

Caffeine


and tea [195,196]



mRNAs

Curcumin






cancer

Apigenin


and celery [203]



splicing

Luteolin


and celery [203]


apigenin

be contained in coffee and tea [195,196] modulate alternative splicing of p53, andSRSF3 target mRNAs

Curcumin


Resveratrol



red wine [191–194]


of SRSF3 and SMN2

Caffeine


and tea [195,196]



mRNAs

Curcumin






cancer

Apigenin


and celery [203]



splicing

Luteolin


and celery [203]


apigenin

be contained in the roots ofturmeric [197–199]

modulate alternative splicing of PKM2 andvarious mRNAs involved in head and neck

cancer

apigenin


Resveratrol



red wine [191–194]


of SRSF3 and SMN2

Caffeine


and tea [195,196]



mRNAs

Curcumin






cancer

Apigenin


and celery [203]



splicing

Luteolin


and celery [203]


apigenin

be contained in parsley and celery [203] interact with the U2 and U5 snRNP andaffect alternative splicing

luteolin


Resveratrol



red wine [191–194]


of SRSF3 and SMN2

Caffeine


and tea [195,196]



mRNAs

Curcumin






cancer

Apigenin


and celery [203]



splicing

Luteolin


and celery [203]


apigenin be contained in parsley and celery [203] show similar effect with apigenin

6-gingerol


6-gingerol

be contained in ginger

[204] inhibit mRNA processing

6-shogaol



Author Contributions: K.-i.F. conceptualized this manuscript; K.-i.F., T.I., and M.M. collected references and wrote session(s); K.-i.F, M.K., and S.M. revised and completed the final manuscript.

Funding: This work was supported in part by grants-in-aid from JSPS KAKENHI (Grant Numbers 2629053, 17K19232, 19K22280, and 19H02884 to S.M and 19K15807 to K.-i.F). This work was also supported in part by grants-in-aid from The Tojuro Iijima Foundation for Food Science and Technology, Fuji Foundation for Protein Research, and The Kieikai Research Foundation to S.M.

Acknowledgments: The authors would like to thank EDANZ (https://jp-author-services.edanzgroup.com) for the English language proofreading by a native speaker.

Conflicts of Interest: The authors declare no conflict of interest.

References

1. Carey, K.T.; Wickramasinghe, V.O. Regulatory Potential of the RNA Processing Machinery: Implications for Human Disease. Trends Genet. 2018, 34, 279–290.

2. Kim, H.K.; Pham, M.H.C.; Ko, K.S.; Rhee, B.D.; Han, J. Alternative splicing isoforms in health and disease. Pflügers Arch. Eur. J. Physiol. 2018, 470, 995–1016.

3. Wang, E.T.; Sandberg, R.; Luo, S.; Khrebtukova, I.; Zhang, L.; Mayr, C.; Kingsmore, S.F.; Schroth, G.P.; Burge, C.B. Alternative isoform regulation in human tissue transcriptomes. Nature 2008, 456, 470–476.

4. Sterne-Weiler, T.; Weatheritt, R.J.; Best, A.J.; Ha, K.C.H.; Blencowe, B.J. Efficient and Accurate Quantitative Profiling of Alternative Splicing Patterns of Any Complexity on a Laptop. Mol. Cell 2018, 72, 187–200.

5. Porter, R.S.; Jaamour, F.; Iwase, S. Neuron-specific alternative splicing of transcriptional machineries: Implications for neurodevelopmental disorders. Mol. Cell. Neurosci. 2018, 87, 35–45.

6. Kalsotra, A.; Xiao, X.; Ward, A.J.; Castle, J.C.; Johnson, J.M.; Burge, C.B.; Cooper, T.A. A postnatal switch of CELF and MBNL proteins reprograms alternative splicing in the developing heart. Proc. Natl. Acad. Sci. USA 2008, 105, 20333–20338.

7. Dillman, A.A.; Hauser, D.N.; Gibbs, J.R.; Nalls, M.A.; McCoy, M.K.; Rudenko, I.N.; Galter, D.; Cookson, M.R. mRNA expression, splicing and editing in the embryonic and adult mouse cerebral cortex. Nat. Neurosci. 2013, 16, 499–506.

8. Bhate, A.; Parker, D.J.; Bebee, T.W.; Ahn, J.; Arif, W.; Rashan, E.H.; Chorghade, S.; Chau, A.; Lee, J.-H.; Anakk, S.; et al. ESRP2 controls an adult splicing programme in hepatocytes to support postnatal liver maturation. Nat. Commun. 2015, 6, 8768.

9. Will, C.L.; Lührmann, R. Spliceosome structure and function. Cold Spring Harb. Perspect. Biol. 2011, 3, 1–2. 10. Scotti, M.M.; Swanson, M.S. RNA mis-splicing in disease. Nat. Rev. Genet. 2016, 17, 19–32. 11. Bieberstein, N.I.; Oesterreich, F.C.; Straube, K.; Neugebauer, K.M. First exon length controls active

be contained in ginger [204] inhibit mRNA processing

6-shogaol


6-gingerol



6-shogaol



Author Contributions: K.-i.F. conceptualized this manuscript; K.-i.F., T.I., and M.M. collected references and wrote session(s); K.-i.F, M.K., and S.M. revised and completed the final manuscript.

Funding: This work was supported in part by grants-in-aid from JSPS KAKENHI (Grant Numbers 2629053, 17K19232, 19K22280, and 19H02884 to S.M and 19K15807 to K.-i.F). This work was also supported in part by grants-in-aid from The Tojuro Iijima Foundation for Food Science and Technology, Fuji Foundation for Protein Research, and The Kieikai Research Foundation to S.M.

Acknowledgments: The authors would like to thank EDANZ (https://jp-author-services.edanzgroup.com) for the English language proofreading by a native speaker.


References

1. Carey, K.T.; Wickramasinghe, V.O. Regulatory Potential of the RNA Processing Machinery: Implications for Human Disease. Trends Genet. 2018, 34, 279–290.

2. Kim, H.K.; Pham, M.H.C.; Ko, K.S.; Rhee, B.D.; Han, J. Alternative splicing isoforms in health and disease. Pflügers Arch. Eur. J. Physiol. 2018, 470, 995–1016.

3. Wang, E.T.; Sandberg, R.; Luo, S.; Khrebtukova, I.; Zhang, L.; Mayr, C.; Kingsmore, S.F.; Schroth, G.P.; Burge, C.B. Alternative isoform regulation in human tissue transcriptomes. Nature 2008, 456, 470–476.

4. Sterne-Weiler, T.; Weatheritt, R.J.; Best, A.J.; Ha, K.C.H.; Blencowe, B.J. Efficient and Accurate Quantitative Profiling of Alternative Splicing Patterns of Any Complexity on a Laptop. Mol. Cell 2018, 72, 187–200.

5. Porter, R.S.; Jaamour, F.; Iwase, S. Neuron-specific alternative splicing of transcriptional machineries: Implications for neurodevelopmental disorders. Mol. Cell. Neurosci. 2018, 87, 35–45.

6. Kalsotra, A.; Xiao, X.; Ward, A.J.; Castle, J.C.; Johnson, J.M.; Burge, C.B.; Cooper, T.A. A postnatal switch of CELF and MBNL proteins reprograms alternative splicing in the developing heart. Proc. Natl. Acad. Sci. USA 2008, 105, 20333–20338.

7. Dillman, A.A.; Hauser, D.N.; Gibbs, J.R.; Nalls, M.A.; McCoy, M.K.; Rudenko, I.N.; Galter, D.; Cookson, M.R. mRNA expression, splicing and editing in the embryonic and adult mouse cerebral cortex. Nat. Neurosci. 2013, 16, 499–506.

8. Bhate, A.; Parker, D.J.; Bebee, T.W.; Ahn, J.; Arif, W.; Rashan, E.H.; Chorghade, S.; Chau, A.; Lee, J.-H.; Anakk, S.; et al. ESRP2 controls an adult splicing programme in hepatocytes to support postnatal liver maturation. Nat. Commun. 2015, 6, 8768.

9. Will, C.L.; Lührmann, R. Spliceosome structure and function. Cold Spring Harb. Perspect. Biol. 2011, 3, 1–2. 10. Scotti, M.M.; Swanson, M.S. RNA mis-splicing in disease. Nat. Rev. Genet. 2016, 17, 19–32. 11. Bieberstein, N.I.; Oesterreich, F.C.; Straube, K.; Neugebauer, K.M. First exon length controls active

be contained in ginger [204] inhibit mRNA processing

Author Contributions: K.-i.F. conceptualized this manuscript; K.-i.F., T.I., and M.M. collected references andwrote session(s); K.-i.F, M.K., and S.M. revised and completed the final manuscript. All authors have read andagreed to the published version of the manuscript.

Funding: This work was supported in part by grants-in-aid from JSPS KAKENHI (Grant Numbers 2629053,17K19232, 19K22280, and 19H02884 to S.M and 19K15807 to K.-i.F). This work was also supported in part bygrants-in-aid from The Tojuro Iijima Foundation for Food Science and Technology, Fuji Foundation for ProteinResearch, and The Kieikai Research Foundation to S.M.

Acknowledgments: The authors would like to thank EDANZ (https://jp-author-services.edanzgroup.com) for theEnglish language proofreading by a native speaker.


References

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